WO2006132250A1 - Biosensor and biosensor cell - Google Patents

Abstract

Disclosed are a biosensor which enables to form a small biosensor cell having high temperature stability, and a small biosensor cell having high temperature stability. Specifically disclosed is a biosensor (1) comprising a supporting layer (10), a reference electrode layer (11) formed on a front surface (10a) of the supporting layer (10), a working electrode layer (12), a counter electrode layer (13), a temperature sensing means (14), an enzyme membrane (16) covering the surface of the working electrode layer (12), and a heater member (15) formed on a back surface (10b) of the supporting layer (10). Also specifically disclosed is a biosensor cell comprising a sample chamber (21) into or from which a sample liquid flows, and the biosensor. In this biosensor cell, the reference electrode layer (11) and the counter electrode layer (13) are arranged in the sample chamber (21) so that they can be in contact with the sample liquid, the temperature sensing means (14) is arranged in the sample chamber (21) so that it is able to measure the temperature of the sample liquid, and the enzyme membrane (16) covering the working electrode layer (12) is arranged in the sample chamber (21) so that it can be in contact with the components of the sample liquid which are the measurement objects.

Description

 Specification

 Biosensor and biosensor cell technology

 The present invention relates to a biosensor and a biosensor cell, and more particularly to a biosensor capable of forming a small biosensor cell having high temperature stability and a small biosensor cell having high temperature stability.

 Background art

 [0002] A biosensor is a sensor using a biological substance or a biologically related substance (hereinafter referred to as a biological substance) as a molecular identification element. For example, an enzyme sensor using an enzyme as a biological substance or the like is known. ing. The biosensor is used to measure the concentration of a biological substance having mutual affinity with the biological substance used as a molecular identification element, such as a substrate or a coenzyme, in a living body or blood. As the molecular identification element used in the nanosensor, a biological substance or the like having an affinity for it is selected according to the biological substance or the like to be measured. For example, in the case of a biosensor that measures the glucose concentration in a living body or blood (also referred to as a sample solution), glucose oxidase is selected as an enzyme as a molecular identification element.

 [0003] The nanosensor is also used in a continuous measurement device that continuously measures, for example, glucose concentration in blood vessels or subcutaneous tissues of diabetic patients and the like. The biosensor used in this continuous measurement device is required to have a small size, high sensitivity, high stability, and long life.

 [0004] As a conventional biosensor, for example, an electrode having a working electrode and a reference electrode or an outer side of the working electrode is dispersed in a non-crosslinking hydrophilic polymer and a non-crosslinking hydrophilic polymer. And a biosensor comprising an enzyme membrane layer containing a cross-linked enzyme (see Patent Document 1). This biosensor has a cylindrical shape, is small, has excellent sensitivity and stability, and has a long life. However, if it can be further reduced in size, it is more preferable to be a biosensor.

[0005] As a conventional noise sensor that can be made smaller than the cylindrical biosensor, for example, (1) an electrode system comprising at least a measurement electrode and a counter electrode layer provided on an insulating support layer; and (2) the electrode so as to form a space portion facing a part of the measurement electrode and the counter electrode layer. A spacer stacked on top, (3) a reaction reagent portion formed in the space, and (4) a cover plate force superimposed on the spacer, the support layer, the spacer, and the cover. A space surrounded by a plate forms a capillary that is a sample solution passage, and the reaction reagent part includes an oxidoreductase, an electron carrier, a hydrophilic polymer, and a surfactant. A plate-like biosensor has been proposed (see Patent Document 2). Although this biosensor can be downsized, the reaction reagent part dissolves and undergoes an oxidation-reduction reaction each time measurement is performed. There is a need.

 [0006] By the way, an enzyme reaction has an optimum temperature, and the activity of the enzyme changes depending on the temperature. Therefore, there is a problem in that the measurement accuracy of the biosensor decreases because the measurement value changes as the measurement temperature changes. For example, in a neurosensor that uses glucose oxidase, if the measurement temperature fluctuates by 1 ° C, the measurement concentration fluctuates by about 5 mgZdL.

 Therefore, when measuring the concentration of a biological substance or the like in a sample solution using a biosensor, it is necessary to keep the measurement temperature constant. For example, a biosensor is installed in a temperature-controlled room, and the temperature of the temperature-controlled room is adjusted every time it is used. Another possible method is to correct the enzyme activity in the enzyme reaction according to the measured temperature.

 Patent Document 1: Japanese Patent Laid-Open No. 8-5601

 Patent Document 2: Japanese Patent Laid-Open No. 2000-221157

 However, the method for adjusting the temperature of the temperature-controlled room needs to manage the temperature of the entire temperature-controlled room in which the biosensor is arranged, so that the temperature of the biosensor (measured temperature) reaches a constant temperature. For example, it takes more than 30 minutes and there is a problem that preparation time is long before starting measurement. There is also a problem that the device for controlling the temperature of the temperature-controlled room becomes complicated and large.

[0010] On the other hand, the method for correcting the activity of the enzyme has different temperature activity depending on the type of the enzyme, and may not be able to sufficiently cope with the case where the temperature changes every time during the measurement. Not realistic. Therefore, a biosensor that solves these problems is desired. Disclosure of the invention

 Problems to be solved by the invention

 An object of the present invention is to provide a biosensor that can be formed into a small biosensor cell having high temperature stability. Another object of the present invention is to provide a small biosensor cell having high temperature stability. Means for solving the problem

 [0012] As means for solving the above problems,

 Claim 1 includes a support layer, a reference electrode layer, a working electrode layer, a counter electrode layer, and a temperature detection means formed on the front side surface of the support layer, an enzyme film covering the surface of the working electrode layer, A biosensor having a heater member formed on the back side surface of the support layer. Claim 2 is the biosensor according to claim 1, wherein the support layer is a film.

 3. The nanosensor according to claim 1, wherein the heater member is formed on a surface of a substrate, and the support layer formed on the surface of the heater member is a resist insulating layer. A sample chamber into which the liquid flows in and out, the reference electrode layer, and the counter electrode layer are disposed in the sample chamber so as to be in contact with the sample liquid, and the temperature detecting means is provided in the sample chamber so as to be able to measure the temperature of the sample liquid. The biosensor according to any one of claims 1 to 3, wherein an enzyme film disposed and covering the working electrode layer is disposed in the sample chamber so as to be in contact with a component to be measured in the sample solution. Is a biosensor cell. The invention's effect

[0013] According to the biosensor of the present invention, since the temperature detecting means and the heater member are provided on the support layer formed into a layer that can be reduced in size, the working electrode layer in the sample solution and the biosensor is provided. Etc. (hereinafter referred to as “sample solution”) is detected by the temperature detecting means, and based on this, the temperature of the sample solution can be adjusted by the heater member. Therefore, this biosensor can be downsized and the temperature can always be kept constant. According to the biosensor cell of the present invention, since the biosensor of the present invention is provided, it is not necessary to use a complicated and large temperature control device such as a temperature-controlled room. Therefore, the biosensor cell of the present invention can be reduced in size and reduced in size and can be reduced in size by effectively utilizing the size of the small biosensor.

 [0015] Further, according to the biosensor cell of the present invention, since the sample chamber and the biosensor of the present invention are provided, the temperature of the sample solution or the like in the sample chamber can be easily adjusted. Therefore, the temperature of the sample solution can be adjusted to a desired temperature in a short time, and the temperature of the sample solution can be kept constant constantly in response to the temperature change of the sample solution. Therefore, the nanosensor cell of the present invention has high temperature stability and can measure biological substances and the like with high accuracy.

 Brief Description of Drawings

 FIG. 1 is a schematic perspective view showing a biosensor as an example of the present invention, FIG. 1 (a) is a schematic perspective view showing the front side thereof, and FIG. 1 (b) is a back side view thereof. FIG. 2 is a schematic top view showing a biosensor chip capable of forming a biosensor as an example of the present invention.

 FIG. 3 is a schematic top view showing a biosensor chip capable of forming a biosensor as an example of the present invention. FIG. 3 (a) is a schematic top view showing the surface of one support layer, and FIG. ) Is a schematic top view showing the other support layer surface.

 FIG. 4 is a schematic top view showing a front side surface of a biosensor as another example of the present invention.

 FIG. 5 is a schematic cross-sectional view showing a biosensor cell as an example of the present invention.

 FIG. 6 is a schematic top view showing a biosensor cell as an example of the present invention.

 FIG. 7 is a schematic exploded perspective view of a biosensor cell as an example of the present invention.

 FIG. 8 is a partial cross-sectional schematic diagram showing an example of use as an example of a biosensor. FIG. 9 is a schematic cross-sectional view showing a support layer on which an electrode and a heater member are mounted, which is an example of a biosensor.

FIG. 10 is a schematic explanatory view showing an example of a biosensor cell. FIG. 11 is a schematic explanatory diagram showing another example of a biosensor cell.

 1, 2 Biosensor

 5, 6 Biosensor chip

 10 Support layer

 10a, 10b, 10c, 10d, 10e, lOf surface

 11 Reference electrode layer

 12 Working electrode layer

 13 Counter electrode layer

 14 Temperature detection means

 l la, 12a, 13a, 14a, 14b, 15a, 15b

 l ib, 12b, 13b Underlayer

 15 Heater material

 16 Enzyme membrane

 17 Protective film

 20 Biosensor cell

 21 Sample room

 22a, 22b Transport pipe

 23 Upper lid member

 24 Gasket

 24a opening

 25 Lower lid member

 25a Insertion part

 26a, 26b Stopper

 26c stripe

 30 Measurement unit

 31 Temperature controller

30a, 31a, 31b wiring 32 means of transport

 A Wrap line

 40 substrates

 41 Heater member

 42 Support layer

 43 Electrode layer

 50 Biosensor

 51 Biosensor cell

 52 Lower part

 BEST MODE FOR CARRYING OUT THE INVENTION

 [0018] As shown in Figs. 1 (a) and (b), a biosensor 1 as an example of the present invention includes a support layer 10 and a reference electrode layer formed on a front side surface 10a of the support layer 10. 11, working electrode layer 12, counter electrode layer 13 and temperature detecting means 14, enzyme film 16 formed on the surface of working electrode layer 12, and back surface 1 of support layer 10 heater member formed on Ob And have 15. This biosensor 1 has a reference electrode layer 11, a working electrode layer 12, a counter electrode layer 13, a temperature detection means 14, and an enzyme film 16 formed on one surface 10a of one support layer 10 on these surfaces. The heater member 15 is formed on the other surface 10b of the support layer 10.

 In the example shown in FIG. 1, the support layer 10 is formed into a plate shape. This makes it possible to reduce the size of the biosensor. If the size can be reduced, the size and thickness of the support layer 10 are not particularly limited. The material for forming the support layer 10 may be an insulating material, for example, plastics such as polyethylene and polyethylene terephthalate, ceramics, glass, paper, and the like. The support layer 10 of the biosensor 1 is made of polyethylene. The thickness of the support layer 10 made of polyethylene is usually 100 to 250 / ζ πι.

As shown in FIG. 1 (a), on the front side surface 10a of the support layer 10, a rectangular counter electrode layer 13 is located in the vicinity of one end thereof. The size, shape, and the like of the counter electrode layer 13 are not particularly limited. The preferred thickness is adjusted to, for example, 5 to: LOO / zm. The material forming the counter electrode layer 13 is a conductive material, and has a stable potential that does not change even when it comes into contact with the sample liquid. Examples of materials that can be obtained include metals such as aluminum, nickel, copper, platinum, gold and silver, conductive metal oxides such as ITO, and carbon materials such as carbon and carbon nanotubes. Among these, the carbon material is preferable in that the counter electrode layer 13 can be easily formed by screen printing the paste. The counter electrode layer 13 of the biosensor 1 is made of carbon! RU

 On the front side surface 10 a of the support layer 10, a rectangular working electrode layer 12 is positioned adjacent to the counter electrode layer 13. The size, shape and the like of the working electrode layer 12 are not particularly limited, but the suitable thickness is adjusted to, for example, 5 to: LOO / zm. The material for forming the working electrode layer 12 is a conductive material, and any material that can obtain a stable potential without being altered even if it contacts the sample solution. Material is used. The working electrode layer 12 of the nanosensor 1 is made of carbon.

 A rectangular reference electrode layer 11 is located adjacent to the working electrode layer 12 and the counter electrode layer 13 on the front side surface 10 a of the support layer 10. The size, shape, and the like of the reference electrode layer 11 are not particularly limited, but the preferred thickness is adjusted to, for example, 5 to: LOO / zm. The material for forming the reference electrode layer 11 is a conductive material, and any material can be used as long as it has a stable potential that does not change in quality even when it comes into contact with the sample solution. Materials, carbon materials, and materials obtained by combining the metal and a salt of the metal. Among these, the silver / salt / silver power electrode layer surface is preferred because it is difficult to ionize. The reference electrode layer 11 of the biosensor 1 is made of silver, Z salt, or silver.

 Between the support layer 10 and the reference electrode layer 11, the working electrode layer 12 and the counter electrode layer 13, the same as the reference electrode layer 11, the working electrode layer 12 and the counter electrode layer 13 is provided. The base layers l lb, 12b and 13b are formed in a shape and slightly smaller than the size. The material for forming these underlayers l lb, 12b and 13b may be a conductive material, for example, the metal, conductive metal oxide, and carbon material. Among these, low resistance materials such as silver and platinum are preferable.

The reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13 are provided with conductive wires lla, 12 a, and 13 a that communicate with the other end of the support layer 10. Through these conductive wires 11a, 12a and 13a, the reference electrode layer 11, the working electrode layer 12 and the counter electrode Each potential of the layer 13 is guided to the measurement unit 30 (see FIG. 8). These conductors l la, 12a and 13a are preferably formed of the same material and the same thickness as the base layers l lb, 12b and 13b.

 [0025] On the front side surface 10a of the support layer 10, a rectangular temperature detecting means 14 is located adjacent to the reference electrode layer 11 and the working electrode layer 12. The temperature detecting means 14 may be any means that senses the temperature of the sample solution or the like. For example, a thermocouple, a resistance thermometer, a thermistor, etc. can be selected. The size, thickness, and the like of the temperature detection means 14 are not particularly limited, and the shape can be formed into, for example, a bead type, a disk type, a rod type, a thin film type, a chip type, or the like. In the nanosensor 1, a thermistor formed in a thin film mold is selected as the temperature detecting means 14. In general, the thermistor can have a very small shape. For example, in the case of a thin film type thermistor, the thickness can be reduced to 0.15-0.25 mm. Therefore, the use of the thermistor contributes to making the biosensor thinner. The thermistor is also capable of detecting minute temperatures as low as 2Z10000 in practice, making temperature control in this biosensor extremely accurate. The material forming the thermistor may be a semiconductor material, and examples thereof include metal oxides such as iron, nickel, manganese, conoretol, and titanium.

 The temperature detection means 14 is provided with conducting wires 14 a and 14 b that communicate with the other end of the support layer 10. The temperature of the sample liquid sensed by the temperature detecting means 14 is transmitted to the temperature control unit (see FIG. 8) via the conductive wires 14a and 14b. The material for forming the conductive wires 14a and 14b may be a conductive material. Examples thereof include the metal, conductive metal oxide, and carbon material. Among these, a low resistance material such as silver is preferable.

In the biosensor shown in FIG. 1 (a), an enzyme film 16 is formed on the surfaces of the reference electrode layer 11, the working electrode layer 12, the counter electrode layer 13, and the temperature detecting means 14. The The enzyme membrane 16 is a membrane in which an enzyme or an enzyme and a mediator are immobilized. In the present invention, it is sufficient for the enzyme membrane to cover the surface of the working electrode layer 12, and it is not necessary to cover the surfaces of the reference electrode layer 11, the counter electrode layer 13, and the temperature detecting means 14. . In order to be able to accurately detect the temperature, the temperature detecting means 14 is preferably covered with the enzyme film and exposed so as to be in direct contact with the sample solution. Good. However, in the biosensor shown in FIG. In order to make this convenient, the enzyme membrane covers the surfaces of the reference electrode layer 11, the counter electrode layer 13, and the temperature detecting means 14. Even if the enzyme membrane covers the surfaces of the reference electrode layer and the counter electrode layer, the sample solution penetrates or diffuses through the enzyme membrane, so that the sample solution is applied to the reference electrode layer and the counter electrode layer. Substantially contactable.

[0028] The enzyme to be immobilized is selected according to the biological material to be measured. For example, when the biological substance to be measured is alcohol, alcohol oxidase, when the biological substance is glucose — D-glucose oxidase, when the biological substance is cholesterol, cholesterol oxidase, the biological substance Is phosphatidylcholine, urease when the biological substance is urea, uricase when the biological substance is uric acid, and lactate dehydrogenase when the biological substance is lactic acid, Oxalate decarboxylase when the substance is oxalic acid, pyruvate oxidase when the biological substance is pyruvic acid, ascorbate oxidase when the biological substance is ascorbic acid, Sometimes flavin-containing Monookishidaze such biological material and the like are Torimechiruamin is selected. The biosensor chip is configured as a glucose sensor with j8-D-glucose oxidase selected.

 [0029] The mediators to be fixed are acids such as pheucene derivatives, 1,4 monobenzoquinone, tetrathiafulvalene, ferricium ion, hexaxanoiron (III) ion, hexaxanoic acid lithium and methylene blue. There are conductive substances that can be reduced.

 [0030] Examples of the immobilization method for immobilizing an enzyme and Z or mediator (hereinafter referred to as an enzyme or the like) include a carrier binding method, a crosslinking method, a comprehensive method, and the like. The carrier binding method is a method in which an enzyme or the like is bound and immobilized on a water-insoluble carrier, and examples thereof include a covalent bonding method, an ionic bonding method, and a physical adsorption method. The cross-linking method is a method of immobilizing an enzyme by reacting with a reagent (cross-linking agent) having two or more functional groups to cross-link the enzyme and the like. The entrapment method is a method of wrapping an enzyme or the like in a fine lattice such as a gel, or a method of coating the enzyme or the like with a semipermeable polymer film.

[0031] The carrier used in the carrier binding method is not particularly limited as long as it is a water-insoluble polymer material. For example, methinoresenorelose, ethinoresenorelose, hydroxyethinoresenorelose, Examples include polysaccharide derivatives such as carboxymethyl cellulose and cellulose acetate, porous polyurethane, polyvinyl alcohol, metal alginate, sodium polyacrylate, and polyethylene oxide. As the crosslinking agent used in the crosslinking method, any reagent having two or more functional groups may be used. Examples thereof include dartal aldehyde, isocyanate derivatives, bisdiazobenzine and the like. Examples of the polymer compound used in the entrapment method include polyacrylamide, polybulu alcohol and the like.

 [0032] The amount of enzyme to be immobilized is arbitrarily set according to the type and use of the enzyme. In the carrier binding method and the inclusion method, the amount of enzyme contained in the carrier, gel, etc. is similarly set arbitrarily. The amount of the enzyme is, for example, about 0.02 to 4% by mass, preferably about 0.02 to 0.2% by mass, with respect to the total mass of the enzyme membrane to be formed.

 [0033] In the nanosensor 1, the enzyme membrane 16 mixes an enzyme or the like with polybulal alcohol such as PVA-SbQ (for example, manufactured by Toyo Gosei Kogyo Co., Ltd.), and fixes the enzyme or the like in the polybulal alcohol. It is formed by the comprehensive law.

[0034] The PVA-SbQ is obtained by adding a stilbazolium group, which is a photosensitive group, in a pendant form to a completely saponified or partially saponified polyvinyl alcohol, which is a basic polymer, and is a few mol% of the hydroxyl group of the basic polymer, for example 1 ˜5 mol% is polybulal alcohol substituted with the above-mentioned photosensitive group. Here, examples of the photosensitive group include a styrylpyridium group and a styrylquinolium group. Such poly Bulle alcoholic photosensitive polymer compound, for example, part number SPP-H- 13 (poly Bulle alcohol polymerization degree 1700, saponification I匕率88%, SbQ introduction rate 1.3 mol _^0/0) , No. SPP- M- 20 (poly Bulle alcohol having a degree of polymerization of 1200, Keni匕率88%, SbQ introduction rate 2.0 mole _^0/0), No. SPP- L- 30 (polyvinyl - alcohol of polymerization degree 600, saponification rate 70%, SbQ introduction rate 3.0 mole _^0/0), part number SPP -S- 10 (poly Bulle alcohol having a degree of polymerization of 2300, Keni匕率88%, SbQ introduction rate 1.0 mol%), and the like Can be mentioned.

[0035] As a method of immobilizing an enzyme or the like on such a polyvinyl alcohol-based photosensitive polymer compound, first, the enzyme or the like is uniformly dissolved or dispersed in a PVA-SbQ aqueous solution, and the solution or dispersion is smoothed. Cast on a transparent plate and dry. Next, a light source that emits light with a wavelength of 300 to 370 nm (for example, sunlight, fluorescent lamp, chemical lamp, xenon lamp, etc.) Used to irradiate both sides of the formed film with light, and photocrosslink the SbQ group by a dimerization reaction. As a result, a fixed membrane such as an enzyme in which the enzyme or the like is fixed to the polyvinyl alcohol-based photosensitive polymer compound is obtained.

 In the nanosensor 1, a protective film 17 is formed on the surface of the enzyme film 16. The protective film 17 is a film that protects the working electrode layer 12 and the like and allows the biological material to be measured to pass through the working electrode layer 12. The protective film 17 may be a film that functions in this manner. For example, a film formed of the water-insoluble polymer material, or a polymer material such as polycarbonate, polybulal alcohol, cellulose acetate, or polyurethane. And a porous film in which pores having a desired diameter are formed in the film formed by the above method. In order to form pores in the film made of the polymer material, a tracking step of tracking the film formed of the polymer material with high-energy heavy ions and immersing the tracked film in an etching solution It is better to adopt an etching process that forms holes by doing so.

 As shown in FIG. 1B, a rectangular heater member 15 is located on the back side surface 10b of the support layer 10. The heater member 15 heats the sample liquid in the sample chamber 21 via the support layer 10 based on the temperature sensed by the temperature detecting means 14, and maintains the temperature of the sample liquid at a desired temperature. . For example, when j8-D-glucose oxidase is selected as the enzyme, the temperature is maintained at about 37 ° C. by the heater member 15.

 [0038] The size, shape, and the like of the heater member 15 are not particularly limited, but the preferred thickness is adjusted to, for example, 5 to: LOO / zm. The material forming the heater member 15 may be any material that generates heat when energized or the like. Examples thereof include the metal, conductive metal oxide, and carbon material. Among these, a carbon material is preferable.

The heater member 15 is provided with conducting wires 15a and 15b that communicate with the end of the support layer 10. Based on the temperature sensed by the temperature detecting means 14, current is led from the temperature control unit 30 (see FIG. 8) to the heater member 15 via the conductors 15a and 15b, and the heater member 15 generates heat. The material for forming the conductive wires 15a and 15b may be any conductive material, for example, the metal, conductive metal oxide, and carbon material. Among these, a low resistance material such as silver is preferable. [0040] The biosensor 1 as an example of the present invention can be manufactured as follows, for example. First, the support layer 10 is formed into a plate shape having a desired size and thickness by, for example, a molding technique such as injection molding, extrusion molding, or press molding, using the material.

 [0041] Next, a base layer l lb is formed using the material in a pattern corresponding to a pattern in which the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13 are disposed on one surface 10a of the support layer 10. , 12b and 13b. These underlayers are formed by thin film formation techniques such as vapor deposition, sputtering, plating, etching, and printing. These underlayers are preferably formed by a screen printing method. Conductive wires l la, 12a, 13a, 14a extending from the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13 to the other end of the support layer 10 on the surface 1 Oa of the support layer 10 and Each of 14b is formed using the above material. As a method for forming these conductive wires, the above-mentioned thin film forming technique can be mentioned. If these conductors and the underlying layer are formed of the same material, they should be formed simultaneously.

 [0042] On the surface 10a of the support layer 10, each of the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13 is formed by the thin film formation technique using the material. These electrode layers are preferably formed by a screen printing method. Further, the temperature detecting means 14 is formed on the surface 10a of the support layer 10 by using the semiconductor material, for example, by the thin film forming technique. The temperature detecting means 14 may be directly formed on the support layer 10 or the temperature detecting means 14 is previously formed in the thin film type, chip type, rod type, etc., and a conductive adhesive or the like is formed on the support layer 10. Alternatively, the temperature detecting means 14 formed in advance may be bonded together.

 An enzyme film 16 is formed so as to cover the reference electrode layer 11, the working electrode layer 12, the counter electrode layer 13 and the temperature detection means 14. The enzyme film 16 is formed by the dip coating method, the spray coating method, the screen printing method, the dispenser method, etc., using the enzyme immobilized by the enzyme immobilization method.

A protective layer 17 is formed on the surface of the enzyme film 16 with the material. For example, the protective layer 17 may be formed by a dip coating method, a spray coating method, a screen printing method, or the like using the liquid of the water-insoluble polymer material. A hole having a desired hole diameter, such as an electron beam, in a film formed in advance by a polymer material The thin film on which the film is formed is preferably bonded or rolled.

 On the other hand, the heater member 15 is formed on the other surface 10b of the support layer 10 as follows. On the surface 10b of the support layer 10, each of the conductive wires 15a and 15b extending to the end portions thereof is formed by the thin film formation technique using the material. Further, the heater member 15 is formed on the surface 10b of the support layer 10 by using the material and by the thin film forming technique. In this way, the biosensor 1 having the pattern shown in FIGS. 1 (a) and (b) is formed.

 [0046] The biosensor 1 shown in Figs. 1 (a) and (b) is composed of a single support layer 10, but the biosensor of the present invention may have a plurality of support layers. Good. For example, a biosensor formed by using biosensor chips 5 and 6 shown in FIGS. 2 and 3 is an example of a biosensor composed of two supporting layers.

 [0047] The biosensor chip 5 shown in FIG. 2 has the biosensor 1 on one surface 10c delimited by a fold line A formed substantially at the center on one surface of one support layer 10. The reference electrode layer 11, the working electrode layer 12, the counter electrode layer 13, the temperature detection means 14, and the enzyme film 16 (not shown) are formed, and the folded line The heater member 15 is formed in the same pattern as the biosensor 1 on the other surface 10d separated by A. The biosensor chip 5 has the same configuration as that of the biosensor 1, and the underlayers l lb, 12b and 13b, the conductors l la, 12a, 13a, 14a, 14b, 15a and 15b, and the protective layer 17 (not shown) Na! ヽ) Power ^ Each is formed. The biosensor chip 5 is folded in a fold line indicated by the one-dot chain line A in FIG. 2 and the backside surfaces of the support layer 10 are joined together to form a biosensor. The biosensor thus formed includes a support layer 10, a reference electrode layer 11, a working electrode layer 12, a counter electrode layer 13 and a temperature detection means 14 formed on the front side surface 10c of the support layer 10. An enzyme membrane 16 formed on these surfaces and a heater member 15 formed on the back side surface 10d of the support layer 10 are provided.

[0048] The biosensor chip 5 is basically the same as the nanosensor 1 so that the same pattern as the front side surface 10a and the back side surface 10b of the biosensor 1 is formed on one surface of the support layer 10. Manufactured. The conductive wires 15a and 15b are connected to the conductive wires l la, 12a, 13 When a, 14a and 14b and the underlayers l lb, 12b and 13b are formed of the same material, they are preferably formed simultaneously. The biosensor chip 5 thus formed is mountain-folded by a folding line indicated by a one-dot chain line A in FIG. 2, and the back side surfaces of the support layer 10 are joined together to form a nanosensor chip. The The backside surfaces of the support layer 10 may be bonded to each other with, for example, a double-sided tape or an adhesive, or may be engaged with an engagement latch or the like. May be joined together. Thus, when the biosensor 1 is folded in half, the reference electrode layer described above is formed on one surface side of the support layer having a shape that can become the support layer 10 in the biosensor 1 and on the front surface of the biosensor 1. 11. The working electrode layer 12, the counter electrode layer 13, the temperature detecting means 14, and the conductors lla, 12a, 13a, 14a, 14b, or a shape that can be a support layer 10 in the biosensor 1 when folded in half. The conductors 15a and 15b are formed on the other side of the support layer at once by, for example, a printing method, and then folded in half and joined to the back side of the support layer by a simple operation. it can.

The biosensor chip 6 shown in FIG. 3 includes two support layers 10e and 10f. As shown in FIG. 3 (a), the biosensor chip 6 has the same pattern as the biosensor 1 on one surface, and the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 1 3. And the temperature detecting means 14, and a support layer 10e in which the enzyme film (not shown) is formed on the surfaces of the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13, and FIG. As shown in FIG. 4, the support layer 10f on which the heater member 15 is formed in the same pattern as the biosensor 1 is formed on one surface. In addition, the supporting layers 10e and 10f of the biosensor chip 6 have the same configuration as the biosensor 1, and the underlying layers llb, 12b and 13b, the conductors lla, 12a, 13a, 14a, 14b, 15a and 15b, and the protection Layer 17 (not shown! /,) Forces are formed. When the support layers 10e and 10f of the nanosensor chip 6 are joined to each other, that is, the back surfaces thereof, a biosensor is formed. The biosensor thus formed includes a support layer 10, a reference electrode layer 11, a working electrode layer 12, a counter electrode layer 13, and a temperature detection unit 14 formed on the front side surface 10 e of the support layer 10. An enzyme membrane 16 formed on these surfaces and a heater member 15 formed on the back side surface 10f of the support layer 10 are provided. [0050] The supporting layers lOe and lOf of the biosensor chip 6 have the same pattern as the front side surface 10a and the back side surface 10b of the nanosensor 1 on one surface of each supporting layer 10e and 10f. Manufactured basically in the same way as Sensor 1. The support layers 10e and 10f formed in this way are joined to each other on the surface by the joining method to form a biosensor chip.

 [0051] In the biosensor of the present invention, for example, the pattern of the working electrode and the like may be changed as desired. For example, as shown in FIG. 4, the temperature detecting means 14 can be disposed between the reference electrode layer 11 and the working electrode layer 12 and the counter electrode layer 13 on the front side surface 10a of the support layer 10. . Biosensor 2 has the same function as biosensor 1 and is manufactured in the same manner.

 [0052] According to the biosensors 1 and 2, since the temperature detecting means 14 and the heater member 15 are provided on the support layer 10 formed into a layer that can be reduced in size, the temperature of the sample solution or the like can be obtained. Is detected by the temperature detecting means 14, and the temperature of the sample liquid or the like can be adjusted by the heater member 15 based on the detected temperature. Therefore, the biosensors 1 and 2 can be miniaturized and the temperature can always be kept constant.

 [0053] In addition, according to the biosensors 1 and 2, by utilizing their sizes, the structure is simple and the size can be reduced, the temperature is high, and the biological material and the like are highly accurate. A biosensor capable of measuring the above can be formed.

 [0054] The arrangement of the reference electrode layer 11, the working electrode layer 12, the counter electrode layer 13 and the temperature detecting means 14 of the biosensors 1 and 2 is various in addition to the arrangement shown in FIGS. Can be changed. Furthermore, the enzyme membrane 16 of the biosensors 1 and 2 is formed so as to cover the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13, but the enzyme membrane is on the surface of the working electrode layer 12. If it is formed! The temperature detecting means 14 is preferably exposed without being covered with an enzyme membrane. This is because the temperature can be accurately detected.

The nanosensors 1 and 2 are used for measuring the concentration of a biological substance or the like in a sample solution. The form is not particularly limited. For example, the form may be incorporated into a batch measurement device such as a portable biosensor device, or may be incorporated into a continuous measurement device such as an artificial spleen device. Yes.

 [0056] Hereinafter, a biosensor cell incorporating the biosensor of the present invention will be described. As shown in FIGS. 5 and 6, a biosensor cell 20 as an example of the present invention includes a sample chamber 21 and a biosensor 1 arranged at a predetermined position. As shown in FIG. 6, the predetermined position where the biosensor 1 is arranged is that of the reference electrode layer 11, the counter electrode layer 13, the temperature detection means 14, and the working electrode layer 12 in the biosensor 1. If the enzyme membrane 16 covering the surface is located in the sample chamber 21,

 [0057] The biosensor cell 20 includes transport pipes 22a and 22b through which the sample liquid flows in and out, an upper lid member 23, a gas get 24, a lower lid casing 25, fasteners 26a and 26b, The sample chamber 21 including the sensor 1 is formed by the upper lid member 23, the gasket 24, and the biosensor 1.

 [0058] The lower lid member 25 is formed in a plate shape, and a loading portion 25a into which the biosensor 1 is loaded is formed. The insertion portion 25a has the same size as the biosensor 1, and the depth is adjusted to be slightly shallower than the thickness of the biosensor 1. The material forming the lower lid member 25 may be any material having heat insulation properties, and examples thereof include plastic and glass. When the lower lid member 25 is formed of a material having heat insulation properties, the temperature around the biosensor is shut off and the temperature of the biosensor cell can be easily controlled. Neusen Sacel 20! / It is made of polycarbonate!

 As shown in FIG. 7, the gasket 24 is formed as a sheet member having an opening 24 a that forms the side wall of the sample chamber 21. The gasket 24 is placed on the lower lid member 25 and the biosensor 1 to form a side wall of the sample chamber 21 and to seal the sample chamber 21. The material for forming the gasket 24 is not particularly limited, and examples thereof include flexible plastic and rubber. The biosensor cell 20 is made of silicone rubber.

As shown in FIG. 5, the upper lid member 23 is placed on the gasket 24 and constitutes the ceiling surface of the sample chamber 21. The upper lid member 23 is formed in a plate shape, and is pierced so that a transport pipe hole into which the transport pipes 22a and 22b are inserted penetrates the upper lid member 23. The material forming the upper lid member 23 is not particularly limited, and is formed of the same material as the lower lid member 25. [0061] The transport pipes 22a and 22b are inserted into the transport pipe holes provided in the upper lid member 23, and allow the sample liquid containing the biological material to be measured to flow into the sample chamber 21 and out of the sample chamber 21. Let The material forming the transport pipes 22a and 22b is not particularly limited, and examples thereof include plastic, rubber, glass, and metal.

 The stoppers 26a and 26b are attached to a laminate of the lower lid member 25, the biosensor 1, the gasket 24, and the upper lid member 23, and hold the laminate so as to seal the sample chamber 21. As shown in FIGS. 5 and 7, in the biosensor cell 20, the fasteners 26a and 26b are formed in a U-shaped lid shape and attached to the laminate from the extending direction of the biosensor 1. It is done. Therefore, a slit 26c that penetrates the biosensor 1 is formed in the stopper 26b. The material forming the fasteners 26a and 26b is not particularly limited as long as it is a material having a strength capable of holding the laminate, and examples thereof include plastics and metals.

 [0063] The biosensor cell 20 is manufactured as follows. First, the transport pipes 22a and 22b, the upper lid member 23, the gasket 24, the lower lid member 25, and the fasteners 26a and 26b are formed. The upper lid member 23 and the lower lid member 25 are formed into a plate shape having a desired size and thickness by using a molding technique such as injection molding, extrusion molding, or press molding, using the above materials. A hole for a transport pipe is drilled in the upper lid member 23 so as to penetrate the upper lid member 23, and a loading portion 25 a for loading the biosensor 1 is formed in the lower lid member 25. The gasket 24 is formed from the above material into a sheet shape having an opening 24a by the forming technique or the like. The transport pipes 22a and 22b are formed using the above materials. The stoppers 26a and 26b are formed of the above-mentioned material into a U-shaped lid shape by the molding technique or the like. A slit 26c for penetrating the noise sensor 1 is formed in the stopper 26b.

[0064] As shown in FIG. 7, the biosensor 1 is inserted into the insertion portion 25a of the lower lid member 25 formed as described above, and then the gasket 24 is placed. At this time, as shown in FIG. 6, the reference electrode layer 11, the counter electrode layer 13, the temperature detecting means 14, and the enzyme film 16 in the biosensor 1 are present in the opening 24a of the gasket 24. Next, adjust the position of biosensor 1 and gasket 24. Next, the transport pipes 22 a and 22 b are inserted into the transport pipe holes of the upper lid member 23. The upper lid member 23 is placed on the gasket 24 and pressed in the stacking direction so that the sample chamber 21 is hermetically sealed. The stoppers 26a and 26b are inserted into both ends of the extending direction from the extending direction to hold the laminate.

 As shown in FIG. 8, a biosensor cell 20 incorporated in a continuous measurement device such as an artificial spleen device can be cited as an example of use of this nanosensor cell 20. The transport tube 22a in the biosensor cell 20 is provided in a collection unit (not shown) for collecting a sample solution, such as a catheter inserted into a patient's vein, a tank for diluting and storing blood collected from the patient, and the like. The other transport pipe 22b is connected to a waste liquid tank (not shown), and transport means 32 such as a pump is interposed in the transport pipe 22b. This transport means 32 allows the sample liquid to flow into and out of the sample chamber 21.

 [0066] The conducting wires lla, 12a and 13a of the nanosensor 1 are connected to the measuring unit 30 via the wiring 30a connected at the end of the support layer 10, respectively. The conducting wires 14a and 14b of the biosensor 1 are connected to the temperature control unit 31 via the wiring 31a connected at the end of the support layer 10, respectively. The conducting wires 15a and 15b of the biosensor 1 are connected to the temperature control unit 31 via wiring 3 lb connected at the end of the support layer 10, respectively. The measuring unit 30 has a potentiostat function for electrochemical measurement. Further, the temperature control unit 31 receives the signal of the temperature detection means 14, and adjusts the current to the heater member 15 based on this signal so as to keep the temperature of the sample liquid or the like at a desired temperature. .

 [0067] In this way, the nano sensor cell 20 is incorporated into a continuous measurement device and used so that continuous measurement is possible. The operation of the continuous measurement apparatus incorporating the biosensor cell 20 will be described.

 [0068] A sample solution for warming up, for example, physiological saline or the like is prepared in the collection unit, the pump 32 is activated, and the sample solution force for warming up is according to the path indicated by arrows B1 to B5 in FIG. The sample chamber 21 flows in and out and is transported to a waste liquid tank (not shown).

[0069] When the warming-up sample solution is transported in this manner, the measurement unit 30 is activated and a constant measurement potential based on the reference electrode layer 11 is applied to the working electrode layer 12. At the same time, the temperature detection means 14 in the sample chamber 21 senses the temperature of the sample liquid and the like, and the signal is transmitted to the temperature control unit 31. Based on the signal sent from the temperature detection means 14 Thus, when it is determined that the temperature of the sample solution or the like is lower than a predetermined temperature, for example, less than about 37 ° C. in the artificial spleen device, a current flows from the temperature control unit 31 to the heater member 15. Then, the heater member 15 generates heat and heats the sample solution in the sample chamber 21 via the support layer 10 of the biosensor 1. At this time, since the temperature detecting means 14 always senses the temperature of the sample liquid and transmits the signal to the temperature control unit 31, the temperature of the sample liquid heated by the heater member 15 reaches a predetermined temperature. The current flowing from the temperature control unit 31 to the heater member 15 is stopped. In this way, during the measurement, the temperature detection means 14 always senses the temperature of the sample solution and transmits the signal to the temperature control unit 31, and the temperature control unit 31 receives the signal transmitted from the temperature detection unit 14. Based on the above, the necessity of heating is determined, and when heating is necessary, a current is passed through the heater member 15. Therefore, the temperature of the sample solution is constantly maintained at a predetermined temperature while the sample solution is being transported.

 As described above, the biosensor cell 20 has a sample solution in the sample chamber 21 by the temperature detecting means 14 and the heater member 15 formed on the surface of the support layer 10 in the biosensor 1. The temperature is adjusted easily by heating these to adjust these temperatures to the desired temperatures. Therefore, the temperature of the sample solution can be adjusted to the desired measurement temperature in a short time, and the temperature of the sample solution can be kept constant at any time by responding quickly to the temperature change of the sample solution. . Therefore, the biosensor cell 20 has high temperature stability and can measure biological substances and the like with high accuracy.

[0071] Next, a glucose measurement mechanism using this continuous measurement apparatus will be described. In order to measure the concentration of darose in the sample solution, first, the sample solution transported from the sampling part is introduced into the sample chamber 21. Then, due to the catalytic action of | 8-D-glucosoxidase, which is formed on the working electrode layer 12 and fixed to the enzyme membrane 16 disposed in the sample chamber 21, the glucose force in the sample solution is also increased between darconoraton and peracid.匕 Hydrogen is produced. When the surface of the enzyme membrane 16 is covered with the protective film 17, the glucose in the sample solution reaches the enzyme membrane 16 through the pores of the protective film 17, and the catalytic action causes darconoraton and hydrogen peroxide. Produces. The produced hydrogen peroxide is decomposed into water and oxygen in the working electrode layer 12. As a result, a current proportional to the glucose concentration flows between the working electrode layer 12 and the counter electrode layer 13. By measuring this current value, the dull in the sample solution is indirectly measured. The course concentration can be calculated.

 [0072] The transport pipes 22a and 22b in the biosensor cell 20 are provided on the upper lid member 23, but may be provided on the gasket 24, for example. In addition, the biosensor cell 20 uses a force that can be held by a pressing member such as a clip, for example, a force that is held by the stoppers 26a and 26b formed in a U-shaped lid shape. Instead, the upper lid member 23 and the lower lid member 25 may be held by providing engagement means such as an engagement latch and engaging them. Further, the biosensor cell 20 is formed in a substantially rectangular parallelepiped shape in which a plate-like upper lid member 23, a sheet-like gasket 24, and a plate-like lower lid member 25 are laminated, but the upper lid member 23, the gasket 24, and Other shapes such as a cylindrical shape may be formed by changing the shape or the like of the lower lid member 25.

 [0073] In the use example as an example of the biosensor cell 20 shown in Fig. 8, the biosensor cell 20 is incorporated in a continuous measurement device such as an artificial spleen device, but a batch such as a simple measurement device or a portable measurement device. You may incorporate in a type | formula measurement apparatus etc. Moreover, these continuous measurement devices, batch-type measurement devices, and the like are not limited to artificial spleen devices.

 [0074] While the preferred embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. Other preferred forms of the invention include the following forms.

 [0075] In the biosensor according to the present invention, the support layer may be a film or a plate. Further, the support layer may be flexible or rigid. For example, when the support layer takes the form of a film having a thickness of 100 to 250 m, the electrode layer formed on one side surface of the support layer can be brought close to the heater member formed on the other side surface. The temperature on the side can be measured more accurately.

 Further, as shown in FIG. 9, the support layer may be formed by laminating a heater member 41, a support layer 42, and an electrode layer 43 in this order on the surface of a substrate 40 formed of a material having rigidity. it can. In this case, the support layer 42 is an insulating material and can be formed as a resist layer by a photoetching technique.

When the support layer is a rigid film, as shown in FIG. 10, the upper lid member 23, the gasket 24, and the biosensor 50 may be overlapped to form the biosensor cell 51. This support layer can hold its own shape like a film 11, the biosensor cell 51 can be formed by superimposing the upper lid member 23, the gasket 24, the biosensor 50, and the lower member 52, as shown in FIG. .

 Example

 The following examples are experimental examples of the present invention. The present invention is not limited in any way by these experimental examples.

 (Example 1)

 Using polyethylene, the support layer 10 was formed into a plate shape having a length of 40 mm, a width of 8.5 mm, and a thickness of 0.25 mm. Next, using a silver paste based on polyester-based rosin, on one surface of the support layer 10, a conductor l la having a thickness of 10 / zm, so as to have the pattern shown in FIG. 12a, 13a, 14a, 14b, 15a and 15b, and underlayers l ib, 12b and 13b having a thickness of 10 m were formed by screen printing, respectively. The width of each conductor was 250 / z m. The formed underlayer l ib has a length of 2.5 mm, a width of 2.5 mm, and a thickness of 10 / zm, and the underlayer 12b has a length of 0.7 mm, a width of 0.7 mm, and a thickness of 10 m. The formation 13b was 9.5 mm long, 2.5 mm wide, and 10 μm thick.

[0079] Next, using a carbon paste based on a mixture of polyurethane resin and salt resin resin, a working electrode layer 12 and a counter electrode layer 13 on the base layers ib and 13b, and The heater member 15 was formed by screen printing, and the reference electrode layer 11 was formed on the base layer ib by screen printing using a silver Z salty silver paste. The formed reference electrode layer 11 has a length of 3 mm, a width of 3.25 mm, and a thickness of 14 / zm, and the working electrode layer 12 has a length of 1.2 mm, a width of 1.2 mm, and a thickness of 20 / zm. Layer 13 was 10 mm long, 3.25 mm wide and 20 μm thick, and heater member 15 was 28 mm long, 5 mm wide and 20 μm thick.

Next, a commercially available thermistor (manufactured by Ishizuka Electronics Co., Ltd., model number 36 4FT) was adhered to the support layer 10 with a conductive adhesive as the temperature detecting means 14.

! [0081] Next, in a 4 mass _^0/0 glucose O Kishida over peptidase (manufactured by Amano Enzaimu Ltd.) PVA - SbQ (Toyo Gosei Co., Ltd., product number SPP- H- 13) and are uniformly mixed to The resulting mixed solution was cast on the surfaces of the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13. Thereafter, the coating film was dried and irradiated with light having a wavelength of 300 to 370 nm to obtain an enzyme film having a thickness of 20 m. In FIG. 1, an enzyme membrane 16 covers the reference electrode layer 11, the working electrode layer 12, the counter electrode layer 13, the temperature detecting means 14, and the conductors l la, 12a, 13a, 14a, 14b, 15a and 15b. In this invention, it is sufficient that the enzyme membrane covers at least the surface of the working electrode layer. However, in order to form the enzyme membrane easily, in this embodiment, as described above. Further, the enzyme film 16 covered the surfaces of the reference electrode layer 11, the working electrode layer 12, and the counter electrode layer 13. Next, the enzyme membrane 16 that covers the reference electrode layer 11, the enzyme membrane 16 that covers the working electrode layer 12, and the enzyme membrane 16 that covers the counter electrode layer 13 are coated at a predetermined size. A protective porous layer 17 that sufficiently covers the enzyme film 16 was formed by pasting together a commercially available porous polycarbonate film (made by osmonitors Co., Ltd., pore diameter: 0.2 m) having slag and rolling with a rolling roller. The temperature detecting means 14 was exposed without being covered with the protective layer 17.

[0082] Next, a biosensor cell 20 incorporating the biosensor 1 manufactured as described above was manufactured.

 [0083] First, an upper lid member 23 and a lower lid member 25 having a length of 36 mm, a width of 17 mm, and a thickness of 8 mm were molded from polycarbonate by an injection molding method. A hole for a transport pipe was drilled in the upper lid member 23. The lower lid member 25 was formed with a loading portion 25a for loading the biosensor 1. Transport pipes 22a and 22b were also prepared. Furthermore, a gasket 24 having a length of 29 mm, a width of 6 mm, and a thickness of 1 mm was formed using silicone rubber, and an opening 24 a having a size of 27 mm × 4 mm was provided. The fasteners 26a and 26b were formed using stainless steel, and a slit 26c was formed in the fastener 26b.

Next, the biosensor 1 is inserted into the insertion portion 25a of the lower lid member 25, and the reference electrode layer 11, the counter electrode layer 13, the temperature detection means 14, and the enzyme film 16 in the biosensor 1 are inserted. The gasket 24 was placed in such a manner that it was exposed in the opening 24a of the gasket 24. Further, the upper cover member 23 in which the transport pipes 22a and 22b are inserted and fixed in the transport pipe hole of the upper cover member 23 is placed on the gasket 24 and pressed in the stacking direction so that the sample chamber 21 is sealed. In this state, the stoppers 26a and 26b were inserted into both ends of the extending direction from the extending direction of the biosensor 1. (Temperature stability test)

 The biosensor of Example 1 was incorporated into the artificial spleen device shown in FIG. 8, and the temperature stabilization time for the measurement temperature to stabilize was measured. This test was repeated 10 times, and the average temperature stabilization time was evaluated. As a result, the average temperature stabilization time is very short, about 5 minutes.

Claims

The scope of the claims  [1] a support layer;  A reference electrode layer, a working electrode layer, a counter electrode layer and a temperature detection means formed on the front side surface of the support layer;  An enzyme membrane covering the surface of the working electrode layer;  A biosensor having a heater member formed on the back side surface of the support layer.  [2] The biosensor according to [1], wherein the support layer is a film.  [3] The biosensor according to [1], wherein the heater member is formed on a surface of a substrate, and the support layer formed on the surface of the heater member is a resist insulating layer. [4] A sample chamber through which sample liquid flows in and out,  The reference electrode layer and the counter electrode layer are disposed in the sample chamber so as to be in contact with the sample solution, and the temperature detecting means is disposed in the sample chamber so as to be capable of measuring the temperature of the sample solution. The biosensor cell according to any one of claims 1 to 3, wherein an enzyme film covering the layer is disposed in the sample chamber so as to be in contact with a component to be measured in the sample solution.